Intercellular communication between parasites and with host cells provides mechanisms for parasite development, immune evasion and disease pathology. Bloodstream African trypanosomes produce membranous nanotubes (NTs) that originate from the flagellar membrane and disassociate into free extracellular vesicles (EVs). Trypanosome EVs contain several flagellar proteins that contribute to virulence in the mammalian host including calflagin, adenylate cyclase (GRESAG4), glycosylphosphatidylinositol phospholipase C (GPI-PLC), calreticulin and metacaspase 4. In addition, the human sleeping sickness parasite Trypanosoma brucei rhodesiense produces EVs that contain the serum resistance associated protein (SRA) a virulence factor necessary for human infectivity. We have shown that T. b. rhodesiense EVs transfer SRA to non-human infectious trypanosomes allowing evasion of human innate immunity. Morphological and biophysical studies have shown that trypanosome EVs can also fuse with phosphatidylcholine liposomes, the trypanosome flagellar pocket and mammalian erythrocytes. Trypanosome EV fusion with erythrocytes alters the physical properties of erythrocytes increasing membrane rigidity resulting in rapid erythrocyte clearance and anemia. The proposed studies will extend these initial findings and address several fundamental questions about the function of membrane NTs and EVs. 1) Are EVs produced during trypanosome infection and if so what is the rate of production and does EV cargo change during the course of infection? 2) Do EVs deliver quorum-sensing molecules that trigger differentiation of BF trypanosomes? 3) Does the NT/EV pathway provide an efflux mechanism to rid human sleeping sickness parasites of trypanosome lytic factors (TLF)? 4) Do EVs deliver trypanosome effector molecules that modulate production of the cytokine, tumor necrosis factor-?, in myeloid cells? 5) Does the fusion of trypanosome EVs with host erythrocytes result in erythrophagocytosis and anemia? 6) What trypanosome proteins are necessary for EVs fusion to membranes? Together the proposed studies will result in a better understanding of the role of trypanosome EVs in cell-cell communication and pathogenesis associated by African trypanosomiasis. We anticipate these studies will lead to the development of new diagnostic tools and offer novel strategies to combat anemia in human sleeping sickness and Nagana.